Showing papers on "RAPD published in 2015"

Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers

Abstract: A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l−1) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator’s type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l−1), IAA (0.1 mg l−1) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l−1 IBA + 100 mg l−1 activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l−1 each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum.

Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers.

Abstract: Dendrocalamus strictus popularly known as ‘Male bamboo’ is a multipurpose bamboo which is extensively utilized in pharmaceutical, paper, agricultural and other industrial implements. In this study, in vitro regeneration of D. strictus through nodal culture has been attempted. Murashige and Skoog’s medium supplemented with 4 mg/l BAP was found to be most effective in shoot regeneration with 3.68 ± 0.37 shoots per explant. The effect of Kn was found to be moderate. These hormones also had considerable effect on the shoot length. The highest shoot length after 6 weeks (3.11 ± 0.41 cm) was noted with 5 mg/l BAP followed by 3.07 ± 0.28 cm with 5 mg/l Kn, while decrease in the shoot length was noted with other treatments. The effect of IBA and NAA individually or in combination at different concentrations on rooting was evaluated. The highest number of root (1.36 ± 0.04) was regenerated on full-strength MS medium supplemented with 3 mg/l NAA, while maximum length of 1.64 ± 0.03 cm of roots was recorded with combination of 1 mg/l IBA and 3 mg/l NAA. Tissue-cultured plants thus obtained were successfully transferred to the soil. The clonal fidelity among the in vitro-regenerated plantlets was assessed by RAPD and ISSR markers. The ten RAPD decamers produced 58 amplicons, while nine ISSR primers generated a total of 66 bands. All the bands generated were monomorphic. These results confirmed the clonal fidelity of the tissue culture-raised D. strictus plantlets and corroborated the fact that nodal culture is perhaps the safest mode for multiplication of true to type plants.

Genetic variation and differentiation in tea (Camellia sinensis) germplasm revealed by RAPD and AFLP variation

Abstract: SummaryGenetic variation and resulting variable groupings in tea (Camellia sinensis) and its wild Camelia relatives were assessed using Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphic (AFLP) markers. Numerous polymorphic bands were generated, of which 266 unambiguous ones were scored. The highest level of polymorphism as determined by the expected heterozygosity (Hav) was detected with AFLPs. Three major groups were recognized in the germplasm based on the parsimony method of cluster analysis. Two of the groups corresponded to varieties assamica and sinensis while the third group consisted of a heterogeneous mix of tea cultivars and related wild species. The Taiwan yamacha, unique tea cultivars grown predominantly in the highlands of Taiwan for the production of pan fired semi-fermented (Oolong and Pouchong) tea clustered in this group. Analysis of phenotypic diversity based on the generated RAPD and AFLP profiles revealed that population diversity (H), decreased in the o...

Identification of grapevine cultivars by DNA analyses: Ptfalls of random amplified polymorphic DNA techniques using 10mer primers

Abstract: RAPD (random amplified polymorphic DNA) analysis is thought to be the method of choice for the identification of species and cultivars from crude extracts without previous knowledge of DNA sequences in the genome. Several PCR (polymerase chain reactions) are done with various primers until the electrophoretic separation of the reaction products yields suitable patterns which are not too complicated but still show enough variability to differen­

Intrafamilial, Preferentially Mother-to-Child and Intraspousal, Helicobacter pylori Infection in Japan Determined by Mutilocus Sequence Typing and Random Amplified Polymorphic DNA Fingerprinting

Abstract: Background The infection route of Helicobacter pylori has been recognized to be mainly intrafamilial, preferentially mother-to-child, especially in developed countries. To determine the transmission route, we examined whether multilocus sequence typing (MLST) was useful for analysis of intrafamilial infection. The possibility of intraspousal infection was also evaluated. Materials and Methods Clonal relationships between strains derived from 35 index Japanese pediatric patients, and their family members were analyzed by two genetic typing procedures, MLST and random amplified polymorphic DNA (RAPD) fingerprinting. Results Mostly coincident results were obtained by MLST and RAPD. By MLST, the allele of loci in the isolates mostly matched between the index child and both the father and mother for 9 (25.7%) of the 35 patients, between the index child and the mother for 25 (60.0%) of the 35 patients. Conclusions MLST is useful for analyzing the infection route of H. pylori as a highly reproducible method. Intrafamilial, especially mother-to-children and sibling, infection is the dominant transmission route. Intraspousal infection is also thought to occur in about a quarter in the Japanese families.

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegia evansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Abstract: Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were...

Molecular markers for grape characterization

Abstract: Research Note Five cultivars and 9 Pinot noir clones were used to test the usefulness of RFLP and RAPD markers and determine whether clonal selections could be differentiated.

Efficacy of RAPD, ISSR and DAMD markers in assessment of genetic variability and population structure of wild Musa acuminata colla

Abstract: North east India is considered as one of the major biodiversity hotspots worldwide and centre of origin of several plant species including Musa. Musa acuminata Colla is known to be one of the wild progenitors of cultivated bananas and plantains. Three single primer based DNA marker techniques viz., random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and directed amplification of minisatellites DNA (DAMD) were used for diversity diagnostics among 25 genotypes of wild M. acuminata collected from Meghalaya province of north east India. A total of 58 primers (26-RAPD, 21-ISSR, and11-DAMD) yielded 451 DNA fragments, of which 395 (87.58 %) were found to be polymorphic in nature. The polymorphic information content (PIC) values were almost identical for each marker system. The resolving power of the marker system was found to be highest in RAPD (3.96) whereas ISSR resolved highest marker index (16.39) in the study. Selected amplicon data obtained through single primer amplification reactions were utilized for determination of diversity within and among the populations of M. acuminata. Nei's genetic differentiation (Gst) value (0.451) indicated higher proportion of the genetic variation within the populations which is supported by the AMOVA analysis (88 %). The study provides insight into the efficacy of RAPD, ISSR and DAMD to analyse the genetic variation existing in the wild Musa germplasm, which can further be exploited for quality trait improvement and domestication of such important horticultural crops. The genetic diversity based population structure may shed light on the genetic basis of speciation and evolution of various species within the genus Musa.

RAPD markers in wild and cultivated Vitis vinifera

Abstract: Some Vitis vinifera cultivars and V. vinifera ssp. silvestris individuals have been subjected to the RAPD analysis in order to estimate the genetic diversity existing within this germplasm. 44 decamer primers of arbitrary sequence have been used for PCR and reproducible band profiles have been obtained. The distribution of the individualized polymorphic DNA markers has not turned out to be different in a remarkable way between cultivated and wild grapevines but this RAPD approach provides for some characteristics useful to analyze genetic relationships even within the Vitis vinifera species.

Detection of genetic variation in Ocimum species using RAPD and ISSR markers.

Abstract: There is a lack of information on the molecular characterization of Ocimum species and hence, efforts have been made under the present study to characterize 17 Ocimum genotypes belonging to 5 different species (O. basilicum, O. americanum, O. sanctum, O. gratissimum and O. Polystachyon) through random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. PCR amplification using 20 RAPD primers generated a total of 506 loci, of which 490 (96.47 %) loci were found polymorphic. The PIC value for RAPD ranged from 0.907 (OPF 14) to 0.954 (OPC 11) with an average of 0.937. The ISSR primers generated a total of 238 loci, of them 234 (98.17 %) loci were polymorphic. The PIC value ranged from 0.892 (UBC 808) to 0.943 (ISSR A12) with an average of 0.923. The average Jaccard’s similarity coefficient based on RAPD and ISSR analysis was 0.58 and 0.52, respectively. Clustering pattern of dendrogram generated using the pooled RAPD and ISSR data showed all Ocimum genotypes in their respective species groups at a cutoff value of 0.49 and 0.42, respectively. Many unique species-specific alleles were amplified by RAPD and ISSR markers. In both marker systems, a maximum number of unique alleles were observed in O. sanctum. The results of the present investigation provided valid guidelines for collection, conservation and characterization of Ocimum genetic resources.

Phenotypic and molecular characterization of clinical isolates of Acinetobacter baumannii isolated from Delhi, India.

Abstract: Acinetobacter has gained importance as a multi-drug resistant and hence a difficult to treat pathogen. This study was done to characterize our isolates with respect to drug resistance and presence of beta-lactamases which is a major mechanism of resistance and to type using RAPD and MLST so that comparison of our clones can be made with the existing international clones. 100 isolates recovered from clinical samples from two hospitals in Delhi were tested for their susceptibility against major groups of antimicrobials. The resistant isolates were screened and confirmed phenotypically for presence of ESBL, MBL and AmpC and MBLs also by PCR. The isolates were typed by RAPD and MLST. Out of the 100 isolates, 91, 78 and 2 % were MDR, XDR and PDR respectively. 97, 100 and 85 were screen positive for ESBL, AmpC and MBL respectively. Of these, 38.1 % were confirmed phenotypically to produce ESBL, 99 % produced AmpC and 29.4 % produced MBL comprising of GIM, VIM, SIM and IMP. MLST showed known STs 110, 188, 146, 69, 103, 108 and 194. Eight new STs were encountered. The RAPD showed a high degree of genetic variability among the isolates. Majority of our isolates were MDR, producing one or more types of beta-lactamases. We encountered drug resistant international clones by MLST which are found in other continents there by confirming their spread to Indian sub continent. No data on ST types of other Indian isolates is available in the MLST database and hence comparison is not possible.

Clonal dissemination of multilocus sequence type ST15 KPC-2-producing Klebsiella pneumoniae in Bulgaria

Abstract: A total of 36 consecutive clinical and two fecal-screening carbapenem-resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD- and MLST-types were identified: the dominating MLST-type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC-2, mostly in combination with CTX-M-15, while for one isolate (ST101) the enzymes OXA-48 and CTX-M-14 were found. All KPC-2-producing transconjugants revealed the presence of IncFII plasmid. The OXA-48- and CTX-M-14-producing isolate showed the presence of L/M replicon type. The dissemination of KPC-2-producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA-48 producing ST101 K. pneumoniae in Bulgaria.

Assessment of mercury heavy metal toxicity-induced physiochemical and molecular changes in Sesbania grandiflora L.

Abstract: Mercury (Hg) is one of the major toxic heavy metals because it bioaccumulates and biomagnifies in animal and human bodies via the food chain. To eliminate heavy metal contamination, plants are being used as removal agents of pollutants/toxic chemicals from the environment. The present study was mainly focused on elucidating the potential phytotoxic effects of Hg heavy metal ion exposure on Sesbania grandiflora seedlings. Growth of seedlings was significantly affected (56 %) at 60 mg L−1 Hg concentration. The level of chlorophyll pigment contents was increased in Hg-treated plants compared to the control. Malondialdehyde content and antioxidative enzyme activities were found to be significantly increased by increasing the concentration of Hg exposure up to 40 mg L−1 while slightly decreased at higher doses. The DNA alterations appearing in the random amplified polymorphic DNA (RAPD) profiles of leaf and root tissues following Hg heavy metal exposure included the disappearance of normal DNA bands and the appearance of new bands compared to the untreated controls. This result strongly indicated that genomic template stability was significantly affected by Hg-induced stress in S. grandiflora seedlings. It is concluded that DNA polymorphisms detected by RAPD fingerprinting analysis could be used as potential molecular markers for the evaluation of Hg heavy metal ion-induced genotoxic effects in other plant species.

Comparative assessment of genetic diversity among Indian bamboo genotypes using RAPD and ISSR markers

Abstract: Bamboo is one of the important plant for pulp, paper and charcoal industries. After China, India is the second largest bamboo reserve in Asia. Around the globe, wide genetic diversity of bamboo is present which serves as the base for selection and improvement. DNA based molecular markers appears to be a striking substitute for systematic assessment of the genetic diversity in conservation and genetic improvement of plants. DNA based molecular markers such as RAPD and ISSR were used to assess the genetic diversity in 13 bamboo genotypes. Total 120 RAPD and 63 ISSR primers were tested, of which only 42 polymorphic primers (30 RAPD and 12 ISSR), gave reproducible amplification profile and were used in this study. 30 RAPD primers yielded total 645 amplified fragments, of which 623 were polymorphic, and 20.76 polymorphic bands per primer were observed across 13 genotypes. 12 ISSR primers produced 246 amplified fragments, of which 241 were polymorphic, and 20.08 polymorphic bands per primer was observed across 13 different genotypes. The Jaccard’s coefficient of RAPD, ISSR and pooled RAPD and ISSR dendrograms ranged from 0.26 to 0.83, 0.23 to 0.86 and 0.26 to 0.84 respectively. The present study found the large genetic diversity present between different elite genotypes of bamboo. Such investigation can deliver a well understanding of the available genotypes, which might be further exploited for the paper industry.

Estimation of genetic diversity of commercial mango (Mangifera indica L.) cultivars using RAPD markers

Abstract: SummaryFifty mango cultivars were screened using Randomly Amplified Polymorphic DNA (RAPD) markers with decamer primers of arbitrary sequence. Out of the 80 primers screened, ten were selected which gave 139 clear and bright fragments. A dendrogram based on Jaccard’s co-efficient of similarity implied a moderate degree of genetic diversity among the cultivars used for experimentation. The hybrids which had one parent in common were placed together. In the cluster, alternate bearers and regular bearers formed separate groups, and the members in each group were very closely linked. Another analysis based on Pearson's co-efficient of similarity revealed a high degree of genetic diversity. In both the analyses, ‘Mulgoa’ was found to be very distinct. This study showed clearly that cultivars from South India unveiled maximum diversity and indicated the potential of RAPD markers for the identification and management of mango germplasm for breeding purposes.

Molecular characterization of Dendrobium nobile Lindl., an endangered medicinal orchid, based on randomly amplified polymorphic DNA

Abstract: Dendrobium nobile Lindl., is an orchid species of immense biopharmaceutical and horticultural importance. The knowledge of its existent genetic variations within wild accessions is crucial for formulation of sustainable utilization strategies. It is distributed in a large landscape area and can grow at altitudes ranging from 800 to 2,000 m. Given that Orchidaceae is one of the largest families of flowering plants, reports on their existing genetic diversity are extremely limited. Molecular study on D. nobile can provide valuable information about the genetic diversity level and genetic relatedness of this important medicinal orchid species. Knowledge of genetic structure of a species also provides resources of a species with its current feature and future evolutionary potential. The genetic structure of D. nobile from Northeast India was investigated using randomly amplified polymorphic DNA (RAPD).The PIC value of RAPD primers was 0.74 and Rp values ranged between 6.80 and 13.23. Overall the Shannon Index, Global coefficient of genetic differentiation (G ST), showed that relative genetic diversity of the populations was fairly high. However, the low value of gene flow (Nm = 0.27) was revealed by the RAPD marker. The results of analysis of molecular variance (AMOVA) revealed that variation amongst the populations was significantly higher than within the populations. A combination of UPGMA and STRUCTURE analysis was employed to estimate the genetic relationships of D. nobile germplasm; interestingly, both the methods presented similar grouping pattern with few differences. Results revealed that 60 individuals belonging to six natural populations in Northeast India were clustered into two major groups. The data represented in this study suggested that the RAPD method was a valuable tool for estimation of genetic diversity and genetic relatedness of the D. nobile germplasm. The present findings are useful outcomes for germplasm conservation and formulation of new breeding strategies for this extremely important medicinal orchid species.

Characterization of Vitis vinifera cultivars by random amplified polymorphic DNA markers

Abstract: During the past two years an alternative strategy based on PCR technology has been described (WILLIAMS et al. 1990)and carried out successfully to differentiate cultivars of several plant species (CARLSON et al. 1991; KLEIN-LANKHORS et al. 1991; PARRAN et al. 1991; WILDE et al. 1992). This procedure, named RAPD for Random Amplified Polymorphic DNA, used single decamer primers of arbitrary sequence to amplify DNA fragments of polymorphic length. We have tested this method for the intraspecific characterization of Vitis vinifera and we report here the results obtained on 8 cultivars. D N A e x t r a c t i o n s were performed from clean, young and fully expanded leaves according to DoYLE and DoYLE (1987) with 1 %of polyvinylpyrrolidone (PVP) in the extraction buffer. Extracted DNA was purified using glass-max DNA isolation spin cartridge system purchased by Gibco Brl. Yield of DNA were measured using the HOECHST dye assay method with a TKO 100 minifluorimeter (Hoefer Scientific Instrument)� Random primers were provided by Operon Technologies Inc. and were not further purified. For R A P D a n a l y s i s the protocol reported by WILLIAMS et al. (1990) was followed with minor modifications. Amplification reactions were carried out in 25 f.ll buffer containing 10 mM Tris pH 8.3, 50 mM KC!, 4 mM MgCl2, 200 f!M each of dATP, dGTP, dCTP, dTTP, 0.2 f!M primer and 0.5 unit of Taq Polymerase (CETUS). Amplification was performed in a Perkin Elmer CETUS DNA thermocycler programmed for 45 cycles of 1 min at 94 OC for denaturing, 1 min at 36 T for annealing, 2 min at 72 T for extension. More than 50 decamers of arbitrary sequences have been screened as primers on total DNA of grape cultivars in way to generate polymorphic PCR products. Many of them allowed the amplification of 2-15 DNA fragments sized from 0.1 to 5 kb. Under described standard conditions the electrophoretic patterns were highly reproducible. Among a set of successful primers, some determined complex profiles with numerous and not discrete bands which were difficult to interprete. Others generated simple and readable patterns but only few of them were discriminant between cultivars. The results obtained with two primers OPA 01 (CAGGCCCTTC) and OPA 18 (AGGTGACCGT) are presented in Figure A and B. OPA 01 allowed to obtain five "phenotypes" among 8 tested cultivars. Two pairs, namely Cabernet Sauvignon and Cabernet franc (Figure A, lanes 3 and 4) and Gamay and Chenin (Figure A, lanes 7 and 8), could not be differentiated. The OPA 18 primer gave more numerous amplified products (Figure B). It yielded to the differentiation of the 8 cultivars. However, two of them, Syrah and Chenin (Figure B, lanes 2 and 8) differed only by a weak but constant band (arrow) of their electrophoretic patterns. In fact only cross comparisons between patterns obtained with the two primers OPA 01 and OPA 18 led to a reliable identification of each cultivar. OPA 01 and OPA 18 have also been tested for clones discrimination within the same variety (data not shown) but without success. Experiments with a wide range of decamer primers are in progress. They indicate that each grape variety and certainly some clones within the same cultivar could be characterized by RAPD markers. As already demonstrated for other culti­ vated plants these markers could also be very useful for genetic mapping of Vitis vinifera.

Genetic diversity of Opuntia spp. varieties assessed by classical marker tools (RAPD and ISSR)

Abstract: Opuntia, commonly named “nopal” in Mexico, is an important crop for its agronomical, economical, ecological and cultural value. Furthermore, it is known for its taxonomic complexity. In this paper, we report the genetic variability of 52 Opuntia cultivars with agronomic and economic importance, classified into 12 different species using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. Ten primers, five for each marker type, were selected to assess their ability to detect polymorphisms in this plant accesions/varieties. Both marker systems generated a total of 307 bands, of which 50.8 % were polymorphic with an average of 15.6 polymorphic bands per primer. Thus, we assume that Mexican Opuntia varieties present broad genetic variation. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the K-12 (RAPD) and IS-06 (ISSR) primers were the most informative ones. Clusters obtained from RAPD, ISSR and a combination of both data sets did not match the actual taxonomic classification. On the other hand, the putative varieties currently classified in the same species were not located in the same cluster. Besides, the varieties included in O. ficus-indica, O. albicarpa and O. megacantha showed broad variation but were not well defined into separate clades; these cultivars possibly have common ancestry. The results presented here support our hypothesis about the existence of a smaller number of Opuntia species in accordance with those currently described, but with high intraspecific genetic variation.

Molecular discrimination and identification of some Turkish grape cultivars (Vitis vinifera L.) by RAPD markers

Abstract: The RAPD-based genetic relationships among 17 indigenous grape varieties (Vitis vinifera L.) of Turkey were compared using 22 decamer primers. The genetic relationships among the cultivars concluded from this research are basically related to the origin of the cultivars.

Determination of genetic and epigenetic effects of glyphosate on Triticum aestivum with RAPD and CRED-RA techniques

Abstract: In this study, the most commonly used herbicide, glyphosate, was investigated for its genotoxic effects on the genome of Triticum aestivum. Five different concentrations of the herbicide were used, and alterations to DNA were measured quantitatively based on their RAPD (Randomly Amplified Polymorphic DNA) profiles. The genomic template stability (GTS%) at each concentration was evaluated, and a decrease was observed with increasing glyphosate concentration. Thus the highest concentration was concluded to be the most effective for causing alteration to DNA. Additionally, the coupled restriction enzyme digestion-random amplification (CRED-RA) technique was used to determine an epigenetic mechanism, e.g. DNA methylation. The polymorphic percentages of all concentration were calculated after herbicide applications. When in glyphosate doses compared with control group, all applications of glyphosate observed to consist of methylation. The methylation levels range from 28.3 to 73.9 % (DNA hypermethylation). In conclusion, based on the RAPD and CRED-RA results, glyphosate causes DNA alterations and methylation.

Analysis of Genetic Diversity and Population Structure in Horsegram (Macrotyloma uniflorum) Using RAPD and ISSR Markers

Abstract: Macrotyloma uniflorum (horsegram) is an underutilized warm season pulse crop used as food and fodder in many semi arid regions of the world. In the present study, genetic structure and diversity of M. uniflorum was analysed using RAPD and ISSR markers. Two other species of the genus Macrotyloma namely M. axillare and M. sar-gharwalensis were also included to assess genetic inter-relationships. In total, 25 polymorphic primers amplified 156 fragments ranging in size from 300 to 3000 bp. Primer wise fragments ranged from 2 (OPR-20) to 13 (OPB-5, OPB-12 and OPS-14) with an average of 6.24 fragments per primer. Highest PIC value of 0.499 was recorded for primer OPR-20 and lowest (0.013) for primer OPR-2 with an average of 0.344. STRUCTURE analysis clustered accessions on the basis of their geographic origin and showed the presence of two distinct gene pools. Dendrogram based on Jaccards similarity coefficient also grouped the accessions into two groups and revealed that M. sar-gharwalensis is more distantly related to the cultivated than M. axillare. PCA analysis also confirmed clustering results shown by STRUCTURE. Partitioning of genetic variation using AMOVA revealed 63 % within population variation with only 37 % genetic variation among populations. Few horsegram accessions showed high levels of genetic diversity which can be exploited in crop breeding programmes for its genetic improvement.

A diverse group of halophilic bacteria exist in Lunsu, a natural salt water body of Himachal Pradesh, India.

Abstract: Five halophilic bacterial isolates namely SS1, SS2, SS3, SS5 and SS8 were isolated from soil sediments of Lunsu, a salty water body. All the bacterial isolates showed growth in LB medium containing up to 8.7% NaCl, pH 7–8 and at temperature range of 30–37°C. The bacterial isolates SS1 and SS3 require at least 3.8% NaCl for their growth, indicating their strict halophilic nature. Interestingly, bacterial isolates SS2, SS5 and SS8 but not SS1 and SS3 exhibited growth in medium supplemented with KCl. Accordingly, Na+ and K+ ions were detected at 1.39 and 0.0035%, respectively in Lunsu water. All the bacterial isolates were analyzed by random amplification of polymorphic DNA (RAPD) using four different random primers and produced PCR fragments ranging from 0.1 to 5 kb in size. Phylogenetic tree based on RAPD finger prints showed that SS1 and SS3 formed one group, while SS2 and SS5 formed the second group, whereas SS8 was out group. Sequence analysis of 16S rDNA identified SS1 and SS3 as Halobacillus trueperi, SS2 as Shewanella algae, SS5 as Halomonas venusta, and SS8 as Marinomonas sp. were deposited in GenBank with accession numbers of KM260166, KF751761, KF751760, KF751762 and KF751763, respectively. This is the first report on the presence of diverse halophilic bacteria in the foot hills of Himalayas.